Rat Liver Glutathione Peroxidase Research Articles

Hepatoprotective Effects of Vitamin E Against Malathion-Induced Mitochondrial Dysfunction in Rat Liver

Background: Malathion is an insecticide of the grouping of organophosphate pesticides (OPs), which shows strong insecticidal effects. In addition, vitamin E reacting to cell membrane site may prevent OP-induced oxidative injury. Objectives: The aim of this study was to examine the protective function of vitamin E on toxicity of malathion, by measuring the activities of liver and liver mitochondrial superoxide dismutase (SOD), catalase (CAT), lipid peroxidation (LPO), and glutathione peroxidase (GPx) in rats. Materials and Methods: The mitochondrial viability was determined in liver. Effective doses of malathion (200 mg/kg/day) and vitamin E (alpha-tocopherylacetate [AT]; 15 mg/kg/day) were administered alone or in combination for 14 days. At the end of the experiment, the liver tissue and liver mitochondria of the animals were harvested and examined. Results: In liver tissue, the activity of LPO and CAT was higher in the malathion group in comparison to controls. AT reduced malathion-induced LPO, SOD, CAT, and GPx in rat liver. Coadministration of AT with malathion improved LPO, SOD, and CAT levels in liver as well as CAT and GPx in liver mitochondria. Malathion-induced mitochondria toxicity was recovered by AT. Conclusions: In conclusion, AT measurement can be beneficial for the safety or recovery of malathion-induced toxic injury in liver tissue and liver mitochondria.

The effect of chromium (VI) on the redox homeostasis and the activity of selenoenzymes thioredoxin reductase and glutathione peroxidase in rat liver and kidneys

The effect of chromium (VI) on the redox homeostasis and the activity of selenoenzymes thioredoxin reductase and glutathione peroxidase in rat liver and kidneys

Penultimate selenocysteine residue replaced by cysteine in thioredoxin reductase from selenium‐deficient rat liver

Selenium is an essential micronutrient for humans and animals, and its deficiency can predispose to the development of pathological conditions. This study evaluates the effect of selenium deficiency on the thioredoxin system, consisting of NADPH, selenoprotein thioredoxin reductase (TrxR), and thioredoxin (Trx); and the glutathione system, including NADPH, glutathione reductase, glutathione, and glutaredoxin coupled with selenoprotein glutathione peroxidase (GPx). We particularly investigate whether inactive truncated TrxR is present under selenium-starvation conditions due to reading of the UGA codon as stop. Feeding rats a selenium-deficient diet resulted in a large decrease in activity of TrxR and GPx in rat liver but not in the levels of Trx1 and Grx1. However, selenium deficiency induced mitochondrial Grx2 10-fold and markedly changed the expression of some flavoproteins that are involved in the cellular folate, glucose, and lipid metabolism. Liver TrxR mRNA was nearly unchanged, but no truncated enzyme was found. Instead, a low-activity form of TrxR with a cysteine substituted for the penultimate selenocysteine in the C-terminal active site was identified in selenium-deficient rat liver. These results show a novel mechanism for decoding the UGA stop codon, inserting cysteine to make a full-length enzyme that may be required for selenium assimilation.

Influence of selenium status on activity of phospholipid hydroperoxide glutathione peroxidase in rat liver and testis in comparison with other selenoproteins

Selenium-deficient rats (-Se, fed a Torula yeast-based diet containing no added selenium for 6 weeks) were injected intraperitoneally with up to 50 μg selenium per kg bodyweight (BW) and sacrificed 6 or 12 hr later. Control rats were fed a similar diet with 0.25 mg Se/kg diet added as sodium selenate. Phospholipid hydroper-oxide glutathione peroxidase (phGSH-Px) and cellular glutathione peroxidase (cGSH-Px) activities were determined in liver and testis. Extracellular glutathione peroxidase (eGSH-Px) activity and selenoprotein P level were measured in plasma. Liver phGSH-Px activity in control rats was small in comparison with liver cGSH-Px activity. Much of the phGSH-Px activity measured in liver (especially under -Se conditions) was accounted for by non-specific NADPH oxidation, which was measurable in the absence of any added substrate in the reaction vial, or when a non-reactive substrate analogue was used. Gross activity of liver phGSH-Px fell only to 76% of control values in selenium deficiency and showed little response to selenium injection. Liver cGSH-Px and plasma eGSH-Px activities in -Se rats were reduced to <2% of control values under the same conditions, increasing after selenium injection only to 2 to 3% of control. Selenoprotein P level in plasma fell to 7% of control levels in -Se rats, returning to a maximum of 43% of control by 12 hr after injection of the highest selenium dose. In testis, phGSH-Px and cGSH-Px fell only to 65% and 45% of control values, respectively, and did not increase significantly in response to resupplementation of selenium under the conditions of this experiment. Based on activity levels, phGSH-Px appears to be of greater relevance in testis than liver. Activity of phGSH-Px in either tissue showed little change with selenium status. None of the peroxidases measured responded as strongly to short-term selenium repletion as did selenoprotein P.

Effect of l-penicillamine hydantoin, an analogue of glutathione, on rat liver glutathione peroxidase, reductase and transferase reactions

In soluble fractions prepared from rat liver homogenates, l-penicillamine hydantoin appeared to be, on the basis of SH consumption measurements, a substrate for glutathione peroxidase but not transferase reactions. When glutathione is incubated with rat liver soluble proteins in the presence of penicillamine hydantoin, formation of oxidized glutathione is inhibited. Calculations from Lineweaver Burk plots point out that inhibition by l-penicillamine hydantoin of the peroxide-dependent oxidations of glutathione is mixed, since both apparent K m and V max values are modified. Preincubation of rat liver soluble proteins with l-penicillamine hydantoin led to a progressive inactivation of glutathione peroxidase. The kinetics of this inactivation process with respect to time and inactivator concentration were studied. Inclusion in the preincubation mixture of SH-containing molecules such as dithiothreitol, l-cysteine or glutathione protected the enzyme against inactivation. However, none of these molecules and neither hydantoin, Triton X-100, phenol, nor dialysis could reverse the enzyme from inactivated to activated form. Mitochondrial glutathione peroxidase was inhibited and inactivated by l-penicillamine hydantoin to the same extent as its cytosolic counterpart. Modifications by penicillamine hydantoin of various subcellular markers enzymes (lactate dehydrogenase, N-acetyl β-glucosaminidase, arylsulfatase C, butyryl-CoA dehydrogenase, lauryl-CoA and glycolate oxidases) were of weak amplitude consisting of either inhibition, inactivation or stimulation,

Effect of selenium status on mRNA levels for glutathione peroxidase in rat liver

To determine the effect of Se status on the level of mRNA for Se-dependent glutathione peroxidase (EC 1.11.1.9), rats were fed either a Se-deficient torula yeast diet (less than 0.02 mg Se/kg diet) or a Se-adequate diet (+0.2 mg Se/kg as Na2SeO3) for greater than 135 d. Liver glutathione peroxidase activity was 0.025 for Se-deficient versus 0.615 EU/mg protein for Se-adequate rats. Total liver RNA and polyadenylated RNA were isolated and subjected to Northern blot analysis using a 700 bp DNA probe from cloned murine glutathione peroxidase. Autoradiography showed that Se-deficient liver had 7-17% of the mRNA for glutathione peroxidase present in Se-adequate liver, suggesting that Se status may regulate the level of mRNA for this selenoenzyme.

The effects of selenium and copper deficiencies on glutathione S-transferase and glutathione peroxidase in rat liver.

Selenium (Se) deficiency in rats produced significant increases in the activity of hepatic glutathione S-transferase (GST) with 1-chloro-2,4-dinitrobenzene as substrate and in various GST isoenzymes when determined by radioimmunoassay. These changes is GST activity and concentration were associated with Se deficiency that was severe enough to provoke decreases of over 98% in hepatic Se-containing glutathione peroxidase activity (Se-GSHpx). However, decreases in hepatic Se-GSHpx of 60% induced by copper (Cu) deficiency had no effect on GST activity or concentration. Increased GST activity in Se deficiency has previously been postulated to be a compensatory response to loss of Se-GSHpx, since some GSTs have a non-Se-glutathione peroxidase (non-Se-GSHpx) activity. However, the GST isoenzymes determined in this study, GST Yb1Yb1, GST YcYc and GST YaYa, are known to have up to 30-fold differences in non-Se-GSHpx activity, but they were all significantly increased to a similar extent in the Se-deficient rats.

Phosphoenolpyruvate carboxykinase ferroactivator 1. Mechanism of action and identity with glutathione peroxidase.

A cytosolic protein factor (ferroactivator) facilitates the activation of phosphoenolpyruvate carboxykinase by ferrous ions (Bentle, L. A., and Lardy, H. A. (1977) J. Biol. Chem. 252, 1431-1440). We have extended our studies on the interaction of Fe2+ with this enzyme to establish the conditions under which it is an activator or an inhibitor. Preincubation of phosphoenolpyruvate carboxykinase with Fe2+ and dithiothreitol resulted in irreversible loss of enzyme activity within minutes of Fe2+ addition. This was attributed to an active oxygen species produced by aerobic oxidation of the divalent metal ion in the presence of dithiothreitol as suggested by lack of inhibition in preincubation experiments with Fe2+ under mildly acidic pH; ferroactivation by many H2O2 scavenging enzymes; and lack of inhibition on preincubation under anaerobic conditions. We conclude that Fe2+ per se can activate phosphoenolpyruvate carboxykinase and that ferroactivator protein helps to overcome the deleterious effects of aerobic oxidation. Mechanistic details of ferroactivation and a comparison of the known properties of ferroactivator indicated the similarity of this protein with rat liver glutathione peroxidase. The identity of ferroactivator as glutathione peroxidase was confirmed by the demonstration of catalytic activity, selenium content, and immunological cross-reactivity.

Effect of methylmercury on the activity of glutathione peroxidase in rat liver.

The effect of methylmercury on the activity of glutathione peroxidase in rat liver was studied in vivo. A daily dose of 10mg methylmercuric chloride/kg body weight was administered subcutaneously to 15 male Wistar rats for 10 days, and the glutathione peroxidase activity in the liver was measured to compare with the control activity. A marked decrease was observed in the glutathione peroxidase activity in the experimental animals, which measured as low as 40% in comparison to that in the control animals. It can be speculated that the inhibition of glutathione peroxidase activity plays a significant role in the development of mercury toxicity and that the protective effect of selenium and vitamin E on the mercury intoxication might be partly due to preserving the glutathione peroxidase activity in the antioxidative defense mechanisms.

Development and application of organic reagents for analysis. IX. A sensitive fluorometric method for the determination of glutathione peroxidase activity.

A fluorometric method for the determination of rat liver glutathione peroxidase (GSH-Px) activity has been developed, based on the determination of the concentration of unreacted reduced glutathione in the assay mixture with N-{p-[2-(6-dimethylamino)benzofuranyl]phenyl}maleimide as a fluorogenic reagent. This method is very sensitive and can assay GSH-Px activity in 50 μg wet weight of rat liver. A linear relationship was obtained between GSH-Px activity and amount of sample over the range of 10-100 μg protein. GSH-Px activities obtained by this method correlated well with those obtained by the conventional colorimetric method with 5, 5'-dithiobis(2-nitrobenzoic acid).

Effect of garlic on the hepatic glutathione S-transferase and glutathione peroxidase activity in rat

It was attempted to observe the effect of garlic on the hepatic glutathione s-transferase and glutathione peroxidase activity in this study. Glutathione s-transferase (EC 2.5.1.18) are thought to play a physiological role in initiating the detoxication of potential alkylating agents, inclnding pharmacologically active compounds. Glutathione peroxidase (EC 1.11.1.9) might play an important role in the protection of cellular structures against oxidative challenge. The activities of glutathione s-transferase and glutathione peroxidase in rat liver were increased by the treatment of garlic juice. Allicin fraction, heat-treated allicin fraction and garlic butanol fraction markedly inhibited glutathione s-transferase activityin vitro, whereas glutathione peroxidase activity was significantly increased in heat-treated allicin fraction and garlic butanol fraction.

Influence of mercury (II), cadmium (II), methylmercury, and phenylmercury on the kinetic properties of rat liver glutathione peroxidase.

The effect of phenylmercury and methylmercury on rat liver glutathione peroxidase (GSH X Px) is investigated and compared with that of Hg(II) and with some previously reported results for Cd(II). Analysis of the kinetics of metal binding to the enzyme gives apparent inhibition rate constants: kc = 9.7 mM-1 min-1 for all three mercury compounds and 75 mM-1 min-1 for CdCl2. Glutathione (0.2 mM) protects the enzyme from metal inhibition, decreasing the apparent inhibition rate constants (kc) by 3.6 times for mercury compounds and 4.4 times for CdCl2. KI for the three mercury compounds is found to be 53 microM. It is unexpected that the same value of KI exists for all three forms of mercury studied and that inhibition of the enzyme by the metals is a relatively slow process. For Cd(II) the value of KI is 8.5 microM. It is suggested that inhibition of GSH X Px enzyme activity by cadmium, mercury, and organic mercury salts may not be due to simple complexation of the active site selenium moiety but may be due to a slower process, e.g., an alteration of the enzyme tertiary or quaternary structure.

Amino acid sequence around the active-site selenocysteine of rat liver glutathione peroxidase

Glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) from rat liver was purified to at least 95% purity. A peptide of 16 amino acids, including the active-site selenocysteine (SeCys) residue, was isolated from a tryptic digest by reverse-phase HPLC and gel filtration. The amino acid sequence of the first 46 residues of glutathione peroxidase was obtained from the overlapping sequences of the tryptic selenopeptide and the intact subunit. The selenocysteine residue was located at residue 41, and the sequence of the tryptic selenopeptide was Val-Leu-Leu-Ile-Glu-Asn-Val-Ala-Ser-Leu-SeCys-Gly-Thr-ThrThr-Arg. The sequence was analyzed by computer for homology with other proteins, but no closely related sequences were found. Glutathione peroxidase is the first selenoprotein for which sequence data have been obtained, and the methods described should be applicable to mapping and to sequence analysis of Se-containing peptides from other selenoproteins.

Purification and immunochemical analysis of rat liver glutathione peroxidase.

Glutathione peroxidase (EC 1.11.1.9) was purified about 1500-fold from rat liver cytosol by means of ammonium sulfate fractionation and chromatographies on CM-Sephadex C-50, Sephacryl S-200 and activated thiol-Sepharose 4B. The final enzyme preparation proved to be homogeneous on polyacrylamide gel electrophoresis and to be composed of four identical subunits with a molecular weight of 20,000. The rabbit antiserum against the purified enzyme gave a single fused precipitation line between the crude and purified enzymes. A quite similar displacement curve was observed for both liver cytosols from rats fed 20% casein diets with and without selenium supplementation, as a result of immunochemical titration with the antiserum. It therefore is apparent that an immunochemically recognizable protein other than glutathione peroxidase does not accumulate in a detectable quantity, if there is any in rat liver; dietary selenium-induced increase of glutathione peroxidase activity in the liver is accompanied with protei...

Effect of inducers of drug-metabolizing enzymes on glutathione reductase and glutathione peroxidase in rat liver

Cytosolic glutathione reductase activity of rat liver was shown to increase to about 250% of control values after treatment of the animals by intraperitoneal injections of trans-stilbene oxide. The time course and dose-response relationship of the induction brought about by trans-stilbene oxide were determined. The increase of activity was accompanied by a similar increase of protein precipitable by antibodies to rat liver glutathione reductase. These results strongly indicate that increase of glutathione reductase activity in response to treatment with this inducer is the result of true induction. Phenobarbital and 3-methylcholanthrene increased the activities per mg protein in the cytosol fraction by 80 and 24%, respectively. Glutathione reductase was purified to homogeneity from rats treated with trans-stilbne oxide. The molecular and kinetic properties investigated were not significantly different from those of the enzyme from control animals. Selenium-dependent glutathione peroxidase was not induced in the hepatic cytosol of animals treated with trans-stilbene oxide.

Rapid, High-Yield Purification of Rat Liver Glutathione Peroxidase by High Performance Liquid Chromatography

Abstract Glutathione peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) was purified 3500-fold from rat liver with a yield of 42% using high performance liquid chromatography. The crucial purification step was size-exclusion chromatography on a Spherogel TSK-3000SW column, and the purified enzyme eluted as a single peak. The enzyme stained as a single band following SDS-gel electrophoresis. The molecular weight of the enzyme was estimated to be 105,000, and the subunit molecular weight determined by SDS-gel electrophoresis was 25,000. Polyacrylamide gel electrophoresis indicated five bands of protein with a broad of enzymatic activity. Isoelectric focusing resulted in a peak of enzymatic activity at pH 6.9 with a shoulder at pH 7.3. The specific activity of the purified enzyme was 1,100 μmol of NADPH oxidized per minute per milligram of protein.

Purification and immunohistochemical localization of rat liver glutathione peroxidase

Glutathione peroxidase was purified from the rat liver to give a single protein band in polyacrylamide gel electrophoresis. Rabbits were immunized with this purified enzyme, and a highly specific anti-glutathione peroxidase antiserum was obtained. Using this antibody, an immunohistochemical technique (the indirect method of peroxidase-labeled antibody) was applied to study the localization of the enzyme in the liver cells. On immunohistochemical observation, glutathione peroxidase was localized exclusively in the cytoplasm of hepatocytes, and a stronger ‘immuno-staining’ was exhibited in the peripheries of the hepatic lobules than in the central zone.

Identification of the catalytic site of rat liver glutathione peroxidase as selenocysteine.

A procedure was developed to isolate 75Se-labeled rat liver glutathione peroxidase (glutathione:H2O2 oxidoreductase, EC 1.11.1.9) at 30--50% purity with 20--30% yields in 4--5 days. Using these preparations of glutathione peroxidase, the selenium moiety in the enzyme was identified as selenocysteine by derivatizing the seleno group with either iodoacetate or ethylenimine in the intact protein, hydrolyzing the protein with 6 N HCl, and cochromatographing the 75Se-labeled products with known standards. Techniques employed were anion-exchange chromatography, cation-exchange chromatography, gel-permeation chromatography, two-dimensional thin-layer chromatography, and automated amino acid analysis. The selenocysteine moiety was identified as the catalytic site in glutathione peroxidase by specifically labeling the enzyme with [14C]iodoacetate on the 75Se-labeled selenium atom and fractionating the 14C, 75Se-labeled derivative after acid hydrolysis. It was concluded that the reduced form of glutathione peroxidase contains the selenocysteine selenol (-SeH) at the catalytic site.

Rat liver glutathione peroxidase: Purification and study of multiple forms

The subcellular distributions of glutathione peroxidase and 75Se in rat liver were determined. Approximately 75% of the enzyme and 58% of the 73Se were contained in the cytosolic fraction. Rat liver cytosol glutathione peroxidase was purified 1029-fold from homogenate to yield a sample with a specific activity of 278 μmol of NADPH oxidized/ min/mg of protein. The purified enzyme was subjected to disc-gel and sodium dodecyl sulfate-disc-gel electrophoresis, which confirmed the purity of the enzyme as well as the existence of multiple electrophoretic forms. Glutathione peroxidase existed as a large aggregate after homogenization, and means of dissociating the aggregate were investigated. The enzyme was isolated as a neutrally charged protein and became negatively charged upon storage, a phenomenon that was independent of the aggregation.

Effects of dietary vitamin E on microsomal lipid peroxidation and glutathione peroxidase in rat liver.

Effects of dietary vitamin E on microsomal lipid peroxidation and glutathione peroxidase in rat liver.